Plants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent K m that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDE cd ) at acidic and neutral pH. At neutral pH, VDE cd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin b-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate.

Plants are able to deal with variable environmental conditions; when exposed to strong illumination, they safely dissipate excess energy as heat and increase their capacity for scavenging reacting oxygen species. Both these protection mechanisms involve activation of the xanthophyll cycle, in which the carotenoid violaxanthin is converted to zeaxanthin by violaxanthin de-epoxidase, using ascorbate as the source of reducing power. In this work, following determination of the three-dimensional structure of the violaxanthin de-epoxidase catalytic domain, we identified the putative binding sites for violaxanthin and ascorbate by in silico docking. Amino acid residues lying in close contact with the two substrates were analyzed for their involvement in the catalytic mechanism. Experimental results supported the proposed substrate-binding sites and point to two residues, Asp-177 and Tyr-198, which are suggested to participate in the catalytic mechanism, based on complete loss of activity in mutant proteins. The role of other residues and the mechanistic similarity to aspartic proteases and epoxide hydrolases are discussed.In natural environments, light intensity is variable and often exceeds the saturation limit of photosynthesis (1, 2). As a consequence, excitation energy in excess may lead to production of reactive oxygen species and to oxidative stress, in a process called photoinhibition (2, 3). Photosynthetic organisms have evolved several mechanisms to dissipate excess energy safely and to increase the capacity for scavenging reactive oxygen species. A major role is played by the xanthophyll cycle (4, 5) in which the diepoxide xanthophyll violaxanthin is converted into the epoxide-free zeaxanthin. Zeaxanthin is a key molecule for plant photoprotection, being involved in singlet oxygen scavenging as well as singlet chlorophyll quenching (6 -10).Violaxanthin to zeaxanthin conversion is catalyzed by a lumenal enzyme, called violaxanthin de-epoxidase (VDE).3 The reducing power for the reaction is provided by ascorbate (11), probably in its protonated form (12). VDE is activated when light-driven proton translocation across the thylakoid membrane exceeds the dissipation rate of the proton gradient by ATPase, leading to a decrease in pH in the thylakoid lumen. Inactive VDE is a soluble protein, but upon activation, it associates with the thylakoid membrane (13) where its substrate violaxanthin is located (14). When light intensity decreases, the stromal enzyme zeaxanthin epoxidase converts zeaxanthin back to violaxanthin (15, 16). Both VDE and zeaxanthin epoxidase have been suggested to belong to lipocalins, a multigenic protein family characterized by a conserved structural organization with an 8-strand ␤-barrel (15). VDE and zeaxanthin epoxidase are classified among outlier lipocalins because they do not present all three conserved regions typical of this multigenic family. Because of their rather low similarity with other lipocalins, their true membership of the lipocalin family has been challenged (17). In additio...

Neuroserpin (NS) is a serpin inhibitor of tissue plasminogen activator (tPA) in the brain. The polymerisation of NS pathologic mutants is responsible for a genetic dementia known as familial encephalopathy with neuroserpin inclusion bodies (FENIB). So far, a pharmacological treatment of FENIB, i.e. an inhibitor of NS polymerisation, remains an unmet challenge. Here, we present a biophysical characterisation of the effects caused by embelin (EMB a small natural compound) on NS conformers and NS polymerisation. EMB destabilises all known NS conformers, specifically binding to NS molecules with a 1:1 NS:EMB molar ratio without unfolding the NS fold. In particular, NS polymers disaggregate in the presence of EMB, and their formation is prevented. The NS/EMB complex does not inhibit tPA proteolytic activity. Both effects are pharmacologically relevant: firstly by inhibiting the NS polymerisation associated to FENIB, and secondly by potentially antagonizing metastatic processes facilitated by NS activity in the brain.